Despite the rapid growth of the zebrafish field, an important gap is the ability to easily assess gene function beyond the first zygotic requirement for the gene. Conditional mutations, such as temperature-sensitive mutations that express phenotypes at a high or restrictive temperature (for instance, 33 degrees) but are normal at a low or permissive temperature (24 degrees) allow gene function to be taken away or restored at different times of development, to explore the contribution of gene function in different processes across the animal's life cycle. The zebrafish is ideal for employing temperature-sensitive strategies, because it grows robustly at temperatures from 24 to 33 degrees throughout its life. In this project, we will use forward and reverse genetic screening techniques to evaluate strategies for identifying temperature-sensitive mutations in zebrafish, assemble a panel of 100 temperature-sensitive mutations, and employ secondary screens, including one for phenotypes caused by maternal effects and a second for late larval defects in maintaining adult stem cells. PUBLIC HEALTH RELEVANCE: We will employ forward and reverse genetic screens to identify a panel of 100 temperature-sensitive mutations. We will employ both haploids and diploid screens to identify mutations expressed at 33 degrees, but phenotypically normal at 24 degrees. Secondary screens will include assessing mutants in panel for maternal contribution and for maintaining cycling or quiescent adult stem cells.